CN111621492B - Lactamase and application thereof, and method for preparing (1R,4S) -wenskolide by enzymatic resolution - Google Patents
Lactamase and application thereof, and method for preparing (1R,4S) -wenskolide by enzymatic resolution Download PDFInfo
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- C12P41/00—Processes using enzymes or microorganisms to separate optical isomers from a racemic mixture
- C12P41/006—Processes using enzymes or microorganisms to separate optical isomers from a racemic mixture by reactions involving C-N bonds, e.g. nitriles, amides, hydantoins, carbamates, lactames, transamination reactions, or keto group formation from racemic mixtures
Abstract
The invention discloses a lactamase and application thereof, and a method for preparing (1R,4S) -wenskolide by enzymatic resolution, wherein the amino acid sequence of the lactamase is shown as SEQ ID NO. 1. The nucleotide sequence of the nucleic acid molecule for coding the lactamase is shown as SEQ ID NO. 2. The method for preparing (1R,4S) -wenskolide by enzymatic resolution comprises the following steps: in a buffer solution system, a substrate and the lactamase are mixed to react, and the substrate is 2-azabicyclo [2.2.1] hept-5-en-3-one. The lactamase has the characteristics of high substrate tolerance and high activity, and when the lactamase is used in a method for preparing (1R,4S) -wenskan lactone by enzymatic resolution, a substrate with higher concentration can fully react under the action of the lactamase to obtain the (1R,4S) -wenskan lactone with higher yield and chiral purity, so that the production efficiency is improved, and the production cost is reduced.
Description
Technical Field
The invention relates to the technical field of bioengineering, and particularly relates to a lactamase, application thereof and a method for preparing (1R,4S) -wenskolide by enzymatic resolution.
Background
Gamma-lactams are the compounds 2-azabicyclo [2.2.1]Abbreviation of hept-5-en-3-one (also commonly known as Venus lactone), with molecular formula C6H7NO. One isomer of the gamma-lactam is (-) -gamma-lactam, namely (1R,4S) -wenslilactone, the medicine synthesized from the optically pure (1R,4S) -wenslilactone mainly comprises Abacavir serving as an anti-HIV medicine, and the medicine synthesized from the (1S, 4R) -wenslilactone mainly comprises peramivir serving as an anti-influenza medicine. Therefore, the resolution preparation of (1R,4S) -wenskolide has important economic and social values.
At present, a biocatalytic method is usually adopted to obtain optically pure (1R,4S) -wenskolide, but the tolerance and activity of enzyme currently used for the biocatalytic method are not high generally, so that the concentration of a substrate of a reaction system which can be normally carried out is small, the production efficiency is reduced, and the cost is high.
In view of this, the invention is particularly proposed.
Disclosure of Invention
The invention aims to provide a lactamase, application thereof and a method for preparing (1R,4S) -wenskolide by enzymatic resolution, so as to improve the problems.
The invention is realized by the following steps:
in a first aspect, an embodiment of the present invention provides a lactamase, an amino acid sequence of which is shown in SEQ ID No. 1.
In a second aspect, embodiments of the invention also provide a nucleic acid molecule for encoding the above-described lactamase, optionally having a nucleotide sequence as set forth in SEQ ID No. 2.
In a third aspect, the embodiment of the invention also provides application of the lactamase in preparing (1R,4S) -wenskolide through enzymatic resolution.
In a fourth aspect, embodiments of the present invention also provide a method for preparing (1R,4S) -wenskolide by enzymatic resolution, comprising: in a buffer solution system, a substrate and the lactamase are mixed to react, and the substrate is 2-azabicyclo [2.2.1] hept-5-en-3-one.
At least one of the above embodiments of the present invention has the following advantageous effects: the lactamase of the embodiment of the invention has the characteristics of high substrate tolerance and high activity, and when the lactamase is used in a method for preparing (1R,4S) -wenskolide by enzymatic resolution, a substrate with higher concentration can fully react under the action of the lactamase to obtain (1R,4S) -wenskolide with higher yield and chiral purity, so that the production efficiency is improved, and the production cost is reduced.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the embodiments will be briefly described below, it should be understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and for those skilled in the art, other related drawings can be obtained according to the drawings without inventive efforts.
FIG. 1 is a flow chart of a method for preparing (1R,4S) -wenskolide by enzymatic resolution according to an embodiment of the present invention;
FIG. 2 is a high performance liquid chromatography detection chart of the intermediate product obtained in example 1 of the present invention after reaction of 99% or more;
FIG. 3 is a high performance liquid chromatography detection chart of the target product of example 1 of the present invention after reaction is 99% or more.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.
The invention provides a lactamase and application thereof, and a method for preparing (1R,4S) -wenskolide by enzymatic resolution.
Some embodiments of the invention provide a lactamase, an amino acid sequence of which is shown in SEQ ID No. 1.
SEQ ID NO.1 is:
the inventor aims at obtaining (1R,4S) -Venus lactone by an enzymatic resolution method, and further redesigns an amino acid sequence of the lactamase, and the lactamase has the characteristics of better stereoselectivity, mild reaction conditions, better environmental friendliness and contribution to industrial amplification application. In addition, the lactamase has higher tolerance and activity, can meet the reaction requirement of higher substrate concentration in a reaction system, improves the production efficiency and reduces the production cost.
Some embodiments of the invention also provide a nucleic acid molecule for encoding the above-mentioned lactamase, which has a nucleotide sequence shown in SEQ ID NO. 2.
SEQ ID NO.2 is:
the lactamase can be encoded by the nucleic acid molecule by the conventional genetic engineering method.
Some embodiments of the invention also provide application of the lactamase in preparing (1R,4S) -wenskolide through enzymatic resolution.
Some embodiments of the present invention also provide a method for preparing (1R,4S) -wenskolide by enzymatic resolution, comprising: in a buffer solution system, a substrate and the lactamase are mixed to react, and the substrate is 2-azabicyclo [2.2.1] hept-5-en-3-one. The specific synthetic route is as follows:
specifically, in some embodiments, the reaction temperature of the mixed reaction of the substrate and the lactamase is 15 to 40 ℃, for example, the reaction temperature may be 15 ℃, 20 ℃, 25 ℃, 30 ℃, 35 ℃ or 45 ℃, preferably, the reaction temperature is 20 to 35 ℃, and more preferably, the reaction temperature is 25 to 30 ℃. The specific reaction temperature is selected to be beneficial to better interaction between the lactamase and the substrate, so that a better reaction effect is achieved, and the yield and the purity of a target product are improved.
In order to satisfy the requirement that the lactamase has sufficient effect on the substrate and achieve the purpose of better reaction resolution, the dosage of the lactamase needs to be controlled, and therefore, in some embodiments, the dosage of the lactamase is 1% to 2%, preferably 1%, of the mass of the substrate. When the dosage of the lactamase is too small, the reaction is insufficient, so that the yield and the product purity are reduced, and when the dosage of the lactamase is too large, waste is easily caused, and the production cost is increased.
Further, in order to achieve full utilization of the lactamase enzyme and also to enable the reaction process to proceed fully, in some embodiments, the concentration of the substrate in the buffer solution system is less than 700 g/L. Substrate concentrations above this range tend to cause them to exceed the ability of lactamases to tolerate the enzyme, resulting in reduced yields and product purity. Further, the concentration of the substrate in the buffer solution system is preferably 500-600 g/L, and more preferably 550-600 g/L. The substrate concentration in the range can meet the requirements of the target product on yield and purity even if the substrate concentration is as large as possible in the reaction process.
Further, the buffer solution system in the above embodiment of the present invention is phosphate buffer, preferably, the concentration of the phosphate buffer is 8 to 12mM, for example, the concentration of the phosphate buffer is 10mM, more preferably, the phosphate buffer maintains pH 6 to 8, preferably pH 7, during the reaction. The phosphate buffer solution contains disodium hydrogen phosphate and potassium dihydrogen phosphate, the mass concentration of the disodium hydrogen phosphate in the buffer solution can be 4.328g/L, and the mass concentration of the potassium dihydrogen phosphate in the buffer solution can be 2.65 g/L.
In some embodiments, after the reaction, an organic solvent is added for extraction, preferably the organic solvent is dichloromethane. In other embodiments, the organic solvent is not limited as long as it is a solvent capable of extracting the target product, such as diethyl ether.
In some embodiments, the organic solvent is added and mixed uniformly, and then the mixture is concentrated and then extracted by adding the organic solvent. The organic solvent is added and mixed by stirring, and the stirring time may be 10 minutes or may be adjusted depending on the amount of the solution.
Specifically, in some embodiments, after the reaction is equal to or more than 99%, dichloromethane with a volume of 3 to 5vt% of the reaction system is added, the mixture is uniformly stirred, the mixture is concentrated to a volume of 0.5 to 0.7, for example, to a volume of 3/5, dichloromethane is added for extraction three times, and then the solvent is removed. Wherein, the volume of the dichloromethane used for three times of extraction is 0.9-1.1 times, 0.9-1.1 times and 0.4-0.6 times of the volume of the concentrated solution in sequence, for example, the volume of the dichloromethane used is 1 time, 1 time and 0.5 time of the volume of the concentrated solution in sequence.
The solvent may be removed by evaporating the solvent.
In particular, referring to the flow diagram of fig. 1, some embodiments of the present invention provide a method for preparing (1R,4S) -vinasse lactone by enzymatic resolution, comprising: dissolving a substrate in a buffer solution of 8-12 mM to enable the concentration of the substrate to be 500-600 g/L, adding the lactamase according to 1-2% of the mass of the substrate, reacting at 25-30 ℃, adding dichloromethane of 3-5% of a reaction system after the reaction is more than or equal to 99%, uniformly stirring, concentrating to the volume of 0.5-0.7, adding dichloromethane, extracting for three times, and removing the solvent to obtain the (1R,4S) -wen si lactone.
The features and properties of the present invention are described in further detail below with reference to examples.
The amino acid sequences of the lactamases used in the following examples of the present invention are shown in SEQ ID NO. 1.
Example 1
The method for preparing (1R,4S) -wenskan lactone by enzymatic resolution provided in this example specifically includes the following steps: dissolving 500g of wenscolide in 1L of phosphate buffer solution with the concentration of 10mM, adding 3g of lactamase, starting stirring, reacting at 25 ℃, carrying out intermediate sampling and central control, starting post-treatment after the reaction is more than or equal to 99% (see figure 2), adding 4% of dichloromethane in the reaction system, stirring for 10 minutes, concentrating to the volume of 3/5, extracting for 3 times (1 time, 1 time and 0.5 time) by using 2.5 times of dichloromethane, concentrating a dichloromethane phase after the extraction is finished, recovering a solvent, and evaporating to obtain 135.9g of a target product (1R,4S) -2-azabicyclo [2.2.1] hept-5-en-3-one, wherein the yield is 48.8% and the chiral purity is 100% (see figure 3).
Wherein, FIG. 2 and FIG. 3 are the detection result diagrams of the HPLC detection of the intermediate sample and the final target product after the reaction is more than or equal to 99%.
The reaction central control analysis method comprises the following steps:
the sample processing method comprises the following steps: taking 0.5mL of reaction liquid into a centrifuge tube, adding ethyl acetate for extraction, after layering, absorbing an upper ethyl acetate phase, adding anhydrous sodium sulfate for dehydration, and then sending a sample for detection.
Liquid phase conditions:
column: CHIRALPAK AD-H chiral column
Flow rate: 0.70 mL/min; wavelength: 230 nm; sample introduction amount: 20 mu L of the solution; column temperature: 25 ℃;
mobile phase: n-hexane-isopropanol 90:10,
detecting the liquid phase condition of the product purity:
column: c18250 (250X 4.6mm, 5 μm)
Flow rate: 1.00 ml/min; wavelength: 210 nm; sample introduction amount: 20 mu L of the solution; column temperature: 25 ℃;
mobile phase: 0.1% phosphoric acid acetonitrile 30: 70.
Example 2
The method for preparing (1R,4S) -wenskan lactone by enzymatic resolution provided in this example specifically includes the following steps: dissolving 500g of wenscolide of a substrate by using 10mM 1L of phosphate buffer, adding 3g of lactamase, starting stirring, reacting at 15 ℃, carrying out middle sampling and control, starting post-treatment after the reaction is more than or equal to 99%, adding 4% of dichloromethane of a reaction system, stirring for 10 minutes, concentrating to 3/5 volume, extracting for 3 times (1 time, 1 time and 0.5 time) by using 2.5 times of dichloromethane, concentrating a dichloromethane phase after the extraction is finished, recovering a solvent, and evaporating to dryness to obtain 135.9g of a target product (1R,4S) -2-azabicyclo [2.2.1] hept-5-en-3-one.
Example 3
The method for preparing (1R,4S) -wenskan lactone by enzymatic resolution provided in this example specifically includes the following steps: dissolving 500g of wenscolide of a substrate by using 10mM 1L of phosphate buffer, adding 3g of lactamase, starting stirring, reacting at 20 ℃, carrying out middle sampling and control, starting post-treatment after the reaction is more than or equal to 99%, adding 4% of dichloromethane of a reaction system, stirring for 10 minutes, concentrating to 3/5 volume, extracting for 3 times (1 time, 1 time and 0.5 time) by using 2.5 times of dichloromethane, concentrating a dichloromethane phase after the extraction is finished, recovering a solvent, and evaporating to dryness to obtain 135.9g of a target product (1R,4S) -2-azabicyclo [2.2.1] hept-5-en-3-one.
Example 4
The method for preparing (1R,4S) -wenskan lactone by enzymatic resolution provided in this example specifically includes the following steps: dissolving 500g of wenscolide of a substrate by using 10mM 1L of phosphate buffer, adding 3g of lactamase, starting stirring, reacting at 30 ℃, carrying out middle sampling and control, starting post-treatment after the reaction is more than or equal to 99%, adding 4% of dichloromethane of a reaction system, stirring for 10 minutes, concentrating to 3/5 volume, extracting for 3 times (1 time, 1 time and 0.5 time) by using 2.5 times of dichloromethane, concentrating a dichloromethane phase after the extraction is finished, recovering a solvent, and evaporating to dryness to obtain 135.9g of a target product (1R,4S) -2-azabicyclo [2.2.1] hept-5-en-3-one.
Example 5
The method for preparing (1R,4S) -wenskan lactone by enzymatic resolution provided in this example specifically includes the following steps: dissolving 500g of wenscolide of a substrate by using 10mM 1L of phosphate buffer, adding 3g of lactamase, starting stirring, reacting at 35 ℃, carrying out intermediate sampling and control, starting post-treatment after the reaction is more than or equal to 99%, adding 4% of dichloromethane of a reaction system, stirring for 10 minutes, concentrating to 3/5 volume, extracting for 3 times (1 time, 1 time and 0.5 time) by using 2.5 times of dichloromethane, concentrating a dichloromethane phase after the extraction is finished, recovering a solvent, and evaporating to dryness to obtain 135.9g of a target product (1R,4S) -2-azabicyclo [2.2.1] hept-5-en-3-one.
Example 6
The method for preparing (1R,4S) -wenskan lactone by enzymatic resolution provided in this example specifically includes the following steps: dissolving 500g of wenscolide of a substrate by using 10mM 1L of phosphate buffer, adding 3g of lactamase, starting stirring, reacting at 40 ℃, carrying out intermediate sampling and control, starting post-treatment after the reaction is more than or equal to 99%, adding 4% of dichloromethane of a reaction system, stirring for 10 minutes, concentrating to 3/5 volume, extracting for 3 times (1 time, 1 time and 0.5 time) by using 2.5 times of dichloromethane, concentrating a dichloromethane phase after the extraction is finished, recovering a solvent, and evaporating to dryness to obtain 135.9g of a target product (1R,4S) -2-azabicyclo [2.2.1] hept-5-en-3-one.
The results of the detection calculation of the yield and purity of the target products of the examples 2-6 and the comparison with the example 1 are shown in the table 1.
TABLE 1
| Group of | Temperature of | Yield% | Chiral purity% |
| Example 2 | 15 | 36.8 | 93.8 |
| Example 3 | 20 | 42.5 | 97.3 |
| Example 1 | 25 | 48.8 | 100 |
| Example 4 | 30 | 48.8 | 99.7 |
| Example 5 | 35 | 46.3 | 97.9 |
| Example 6 | 40 | 40.1 | 86.9 |
From the results of Table 1, it can be seen that the yield and chiral purity of the product increased with increasing reaction temperature, reached a maximum at 25-30 ℃ and then showed a significant decrease with increasing reaction temperature.
Examples 7 to 10
The steps and conditions of the enzymatic resolution process for preparing (1R,4S) -vinasse lactone provided in examples 7-10 are essentially the same as in example 1, except that the mass ratio of the added lactamase to the substrate is 0.5%, 1%, 1.5% and 2% in this order.
The results are shown in Table 2, which are obtained by performing the calculation of the yields and purities of the target products of examples 7-10 and counting the results.
TABLE 2
| Group of | The amount of enzyme used | Yield% | Chiral purity% |
| Example 7 | 0.5％ | 45.6 | 98.8 |
| Example 8 | 1％ | 48.6 | 99.9 |
| Example 9 | 1.5％ | 47.8 | 99.8 |
| Example 10 | 2％ | 48.1 | 99.7 |
As can be seen from the results in Table 2, the amount of lactamase used was 1 to 2%, the yield and purity were high, 1.5 to 2.0%, and the yield and purity were not greatly changed even when the amount of lactamase used was increased, so that the amount of lactamase used was 1% as appropriate.
Examples 11 to 16
The steps and conditions of the enzymatic resolution process for the preparation of (1R,4S) -Venus lactone provided in examples 11-16 are essentially the same as in example 1, except that the substrate concentrations are 400g/L, 450g/L, 550g/L, 600g/L, 650g/L and 700g/L, respectively.
The results are shown in Table 3, which are obtained by performing the calculation of the yields and purities of the target products of examples 11 to 16 and comparing the results with example 1.
TABLE 3
As can be seen from the structure in Table 3, the highest concentration of the substrate can be kept at 550-600 g/L, the concentration of the substrate is increased continuously, and the purity and the yield are obviously reduced.
Examples 17 to 19
Examples 17-19 differed from example 1 only in that the product was isolated using the solvents ethyl acetate, diethyl ether, and chloroform in that order instead of dichloromethane, and the final product yields are shown in table 4.
TABLE 4
| Group of | Extraction solvent | Yield of |
| Example 17 | Ethyl acetate | 43.6 |
| Example 1 | Methylene dichloride | 48.8 |
| Example 18 | Ether (A) | 39.6 |
| Example 19 | Trichloromethane | 40.2 |
In each of examples 1 to 19, the pH of the buffer solution was maintained at 7.
Examples 20 to 23
Examples 20 to 23 differ from example 1 only in that the pH of the buffer solution was 5, 6, 8, 9 in this order. The final product was tested for yield and purity and compared to example 1, with the results shown in table 5.
TABLE 5
Comparative examples 1 to 3
Comparative examples 1 to 3 differ from example 1 in that the lactamase of example 1 was replaced with Lipase AK, Lipase Lip ozyme TL IM, lactamase Delftia sp in this order. The final product was tested for yield and purity and compared to example 1, with the results shown in table 6.
TABLE 6
| Group of | Class of enzyme | Yield of | Purity of |
| Comparative example 1 | |
30％ | 78.9％ |
| Comparative example 2 | Lipase Lip ozyme |
25％ | 63.1％ |
| Comparative example 3 | Lactamase Delftia sp | 39％ | 89.8％ |
In conclusion, the embodiment of the invention selects the specific lactamase, can well complete enzymatic resolution of the wensi lactone under the conditions that the temperature is 25-30 ℃, the enzyme dosage is about 1% of the mass of the substrate, and the substrate concentration is less than or equal to 600g/L, obtains the (1R,4S) -wensi lactone with higher yield and purity, has higher substrate tolerance and higher activity compared with the existing method for resolving the wensi lactone by the biological enzyme method, has the highest substrate concentration of 600g/L, improves the production efficiency, reduces the production cost, and has wide application prospect and popularization value.
The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Sequence listing
<110> sequoia anhua biological medicine science and technology Limited
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Ala Thr Ala Thr Asn Ile Leu Gly Ser Gly Val Gly Trp Gly Arg Lys
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ggcagaccgc ctatgaagga ggaaagtccc tacggaagcc tttatgtcat tacggagatc 240
aaagggaagg aagagggtaa actcaagcaa aaaaagatat gtattaaaga taacgtaatg 300
atcgctggca taccaatgct aaatggatct aagatgctgg aagggtttgt gccgaggatg 360
ggtgccactg ttgtctcccg tatttgcgac gaggcaggcg aaatcgtagc gaaaaccaca 420
tgtaaggatt tatgcttctc aggagggtcg gagacgagtt accctgtgcc cgttttgaac 480
ccacgcaatc cgaaatatat ggctggtggc agctcttccg gatcagccgt cgcagtagcg 540
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ttttccatct tcccaactct agaccatctg gggccgatgg caaacaccgt aaaggatgtg 720
gcgttattgc ttgaatacat agctggtaga gacgagctcg attcaaggca acctcagtcg 780
ctacccccac cgcctgttaa accctatagt aagctggacg gcgatgtcaa agacaataag 840
gtaggaattt acaaagaagg gtttaacgtg agcaattctg agaaggatta tgacgaatta 900
gttttggatt ccgccaaaaa gcttgaggac tacggtatca aagtcgaaga tacatcactc 960
ttcctacgtc gcaacggcct ggacataaat acgccagcaa ttgagggagc gactgctacc 1020
aacatcttag ggtcgggtgt aggctgggga cgaaaggggt tgttttggac acaaatagcc 1080
gatttctttg gtaatagtct taaaagccgg gcaagagacc tcccgaacac gtatgaaggc 1140
tacctaatgc tgggatattt aatgattaag atgtacaata acaggtatgt ggcgaaagct 1200
cgtaatttgt ctatcgttct taaggaggcc tacgattccg cactcaaaaa gtatgacgcg 1260
ctaataaacc ctactaccaa aatgaaggct atgcgctaca aatcagaacc cgggttcgat 1320
gagtatttta ttatggccct gggttggatc aataacacag caccattcga cgtcataggc 1380
cacccggcga tgaatattcc tgtaggatac tcggctgggt tacccgtggg tttgatgatc 1440
ataggccgag aaccagagga agataaggtt cttaaactcg ccaacgtctt tcggagaatt 1500
aagaaagcac cgcctacgtt cgcggag 1527
Claims (23)
1. The lactamase is characterized in that the amino acid sequence of the lactamase is shown as SEQ ID NO. 1.
2. A nucleic acid molecule encoding the lactamase of claim 1.
3. The nucleic acid molecule of claim 2, wherein the nucleotide sequence of said nucleic acid molecule is as set forth in SEQ ID No. 2.
4. Use of the lactamase of claim 1 for the preparation of (1R,4S) -wenskan lactone by enzymatic resolution.
5. A method for preparing (1R,4S) -wenskolide by enzymatic resolution is characterized by comprising the following steps: reacting a substrate and the lactamase of claim 1 in a buffer system, wherein the substrate is 2-azabicyclo [2.2.1] hept-5-en-3-one.
6. The method for preparing (1R,4S) -vinassenolide through enzymatic resolution according to claim 5, wherein the reaction temperature is 15-40 ℃.
7. The method for preparing (1R,4S) -vinassenolide through enzymatic resolution according to claim 6, wherein the reaction temperature is 20-35 ℃.
8. The method for preparing (1R,4S) -vinassenolide through enzymatic resolution according to claim 7, wherein the reaction temperature is 25-30 ℃.
9. The method for preparing (1R,4S) -wenskolide through enzymatic resolution according to claim 5, wherein the amount of the lactamase is 1% -2% of the mass of the substrate.
10. The process for preparing (1R,4S) -vinassenolide by enzymatic resolution according to claim 9, wherein the amount of said lactamase is 1% by mass of said substrate.
11. The method for preparing (1R,4S) -Venus lactone by enzymatic resolution according to claim 5, wherein the concentration of the substrate in the buffer solution system is less than 700 g/L.
12. The method for preparing (1R,4S) -vinassenolide through enzymatic resolution according to claim 11, wherein the concentration of the substrate in a buffer solution system is 500-600 g/L.
13. The method for preparing (1R,4S) -vinassenolide through enzymatic resolution according to claim 12, wherein the concentration of the substrate in a buffer solution system is 550-600 g/L.
14. The method for preparing (1R,4S) -wenskan lactone by enzymatic resolution according to claim 5, wherein the buffer solution system is phosphate buffer.
15. The method for preparing (1R,4S) -vinassenolide through enzymatic resolution according to claim 14, wherein the concentration of the phosphate buffer is 8-12 mM.
16. The method for preparing (1R,4S) -vinassenolide through enzymatic resolution according to claim 15, wherein the pH value of the phosphate buffer is kept between 6 and 8.
17. The process for preparing (1R,4S) -vinassenolide by enzymatic resolution according to claim 16, wherein said phosphate buffer is maintained at pH 7.
18. The method for preparing (1R,4S) -wenskan lactone by enzymatic resolution according to any one of claims 5-16, wherein an organic solvent is added for extraction after the reaction.
19. The process for preparing (1R,4S) -vinassenolide by enzymatic resolution according to claim 18, wherein said organic solvent is dichloromethane.
20. The method for preparing (1R,4S) -wenskan lactone by enzymatic resolution according to claim 19, wherein the organic solvent is added and mixed uniformly, and then the mixture is concentrated and then extracted by adding the organic solvent.
21. The method for preparing (1R,4S) -wenskan lactone by enzymatic resolution according to claim 20, which is characterized in that after the reaction is more than or equal to 99%, dichloromethane with the volume of 3-5 vt% of the reaction system is added, the mixture is uniformly stirred and concentrated to the volume of 0.5-0.7, then dichloromethane is added for extraction for three times, and then the solvent is removed.
22. The method for preparing (1R,4S) -vinassenolide through enzymatic resolution according to claim 21, wherein the volumes of dichloromethane used for three times of extraction are 0.9-1.1 times, 0.9-1.1 times and 0.4-0.6 times of the volume of the concentrated solution in sequence.
23. The process for the preparation of (1R,4S) -vinassenolide by enzymatic resolution according to claim 5, comprising: dissolving the substrate in a buffer solution of 8-12 mM to enable the concentration of the substrate to be 500-600 g/L, adding the lactamase according to 1-2% of the mass of the substrate, reacting at 25-30 ℃, adding dichloromethane of 3-5% of a reaction system after the reaction is more than or equal to 99%, uniformly stirring, concentrating to the volume of 0.5-0.7, adding dichloromethane, extracting for three times, and removing the solvent to obtain the (1R,4S) -wen si lactone.
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| CN1203949A (en) * | 1997-06-30 | 1999-01-06 | Basf公司 | Resolution of racemic amino esters by enzyme-catalyzed acylation |
| CN1867677A (en) * | 2003-10-10 | 2006-11-22 | 德古萨股份公司 | Process for preparing enantiomer-enriched alpha-hydroxycarboxylic acids and amides |
| CN107794275A (en) * | 2017-11-06 | 2018-03-13 | 河北省微生物研究所 | The recombinant yeast pichia pastoris of one kind production (+) gamma-lactams enzyme and its construction method and application |
| CN110177790A (en) * | 2016-11-09 | 2019-08-27 | 奥塔万特科学股份有限公司 | The fractionation of optical activity diaza spiro [4.5] decane derivative |
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| US5741657A (en) * | 1995-03-20 | 1998-04-21 | The Regents Of The University Of California | Fluorogenic substrates for β-lactamase and methods of use |
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| CN1203949A (en) * | 1997-06-30 | 1999-01-06 | Basf公司 | Resolution of racemic amino esters by enzyme-catalyzed acylation |
| CN1867677A (en) * | 2003-10-10 | 2006-11-22 | 德古萨股份公司 | Process for preparing enantiomer-enriched alpha-hydroxycarboxylic acids and amides |
| CN110177790A (en) * | 2016-11-09 | 2019-08-27 | 奥塔万特科学股份有限公司 | The fractionation of optical activity diaza spiro [4.5] decane derivative |
| CN107794275A (en) * | 2017-11-06 | 2018-03-13 | 河北省微生物研究所 | The recombinant yeast pichia pastoris of one kind production (+) gamma-lactams enzyme and its construction method and application |
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Denomination of invention: Lactamase and Its Application and Enzymatic Resolution Method for Preparation of (1R, 4S) - Vince Lactone Effective date of registration: 20220929 Granted publication date: 20211026 Pledgee: Suzhou New Area Financing Guarantee Co.,Ltd. Pledgor: Anhui Sequoia Biomedical Technology Co.,Ltd. Registration number: Y2022980016901 |